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Keywords:

  • human embryology;
  • stapedius muscle;
  • pyramidal eminence;
  • pharyngeal arches;
  • Reichert’s cartilage

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

The aim of the study was to systematize the key developmental phases of the stapedius muscle and the pyramidal eminence to clarify their formation, as well as to understand the variations and anomalies that can affect these structures. Sixty human embryos and fetuses between 38 days and 17 weeks of development were studied. The stapedius muscle is formed by two anlagen, one for the tendon, which derives from the internal segment of the interhyale, and another for the belly, located in the second pharyngeal arch medial to the facial nerve and near the interhyale but forming a completely independent anlage. In the interhyale, two segments were differentiated, these forming an angle; at the vertex, the belly of the stapedius muscle is attached. The internal segment is located from the attachment of the belly of the stapedius muscle to the anlage of the stapes, forming the anlage of the tendon of the stapedius muscle. The external segment completely disappears at the beginning of the fetal period. The pyramidal eminence is formed by an anlage independent of Reichert’s cartilage, from the mesenchymal tissue of the tympanic cavity, which condenses around the belly of the stapedius muscle from 12 weeks of post-conception development. The length of the tendon of the stapedius muscle in adults varies, depending on the attachment site of the belly of the stapedius muscle in the interhyale, which would determine the length of the internal segment (anlage of the tendon) and consequently the tendon length. This variation depends on the greater or lesser persistence of the angulation observed during development, between the tendon and the belly of the stapedius muscle.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

The origin and development of the stapedius muscle of the middle ear has been the subject of continual controversy. Different theories have been proposed to explain the development. The classic theory, which appears in embryology textbooks (Hamilton & Mossman, 1975; Corliss, 1979; Abramovich, 1997; Moore & Persaud, 1999; Cochard, 2002; Sadler, 2004), holds that this muscle derives from the second pharyngeal arch without establishing any specifics. The single anlage theory traces the origin from the so-called hyostapedial connection (Anson & Cauldwell, 1942; Anson et al. 1944; Anson & Bast, 1946), from the interhyale (Hanson et al. 1962), or from the remaining connection of the hyoid arch that gives rise to tendon, muscle, and pyramidal eminence (Hough, 1958).

The controversy about the different theories is related in turn to the different conceptions on the organization of the cranial end of Reichert’s cartilage. For the great majority of researchers, the cartilage of the second pharyngeal arch partially or totally forms the stapes of the middle ear (Cauldwell & Anson, 1942; Hamilton & Mossman, 1975; Masuda et al. 1978; Corliss, 1979; Sperber, 1989; Abramovich, 1997; Moore & Persaud, 1999; Cochard, 2002; Sadler, 2004). For others, when the stapes loses its direct continuity with Reichert’s cartilage, the former is joined to the latter by the hyostapedial connection (Anson & Bast, 1946), and it has even been indicated that the cranial end of Reichert’s cartilage would be bifurcated at the interhyale and the laterohyale (Hanson et al. 1962; Nandapalan & Tos, 2000) in such a way that the interhyale would connect with the stapes while the laterohyale would give rise to the pyramidal eminence (Hanson et al. 1962; Nandapalan & Tos, 2000). Other authors theorize on the origin of the pyramidal eminence, indicating that it is formed from Reichert’s cartilage (Hough, 1963).

In previous studies (Rodríguez-Vázquez, 2005; Rodríguez-Vázquez et al. 2006), we determined that the stapes developed at the cranial end of the second branchial arch through an independent anlage of the cartilage of this arch and that the otic capsule is not involved in the formation of the base of the stapes. Furthermore, the so-called laterohyale simply corresponds to the cranial end of Reichert’s cartilage.

The aim of this study was to clarify the controversy concerning the origin of the stapedius muscle and the pyramidal eminence that contains it, in order to establish and systematize the developmental stages of these structures during the embryonic period and the first stages of the fetal period, as these are key to understanding this problem of development of the middle ear. In addition, we also seek to establish the basis for understanding the variations and anomalies that affect these structures, as until now they had been supported on fragmentary knowledge and inconsistent theories.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Twenty embryos and 40 human fetuses from the collection of the Embryology Institute of the Universidad Complutense of Madrid (Spain) were studied. In the embryos, the crown–rump length (CRL) ranged from 10.75 to 31 mm, from 38 to 56 days post-conception development (PCd) (O’Rahilly stages 16–23). In the fetuses, the CRL ranged from 35 to 150 mm (weeks 9–17 PCd). The parameters used to determine gestational age were CRL, weight, and cranial perimeter (O’Rahilly & Müller, 1996). All specimens were obtained from ectopic pregnancies or spontaneous abortions, and no part of the material gave indications of possible malformation. Approval for the study was granted by the Ethics Committee of the Faculty of Medicine of the University Complutense of Madrid.

All specimens were fixed in 10% formalin and embedded in paraffin for processing. Sections were 7–25 µm thick, depending on specimen size. Sections were stained with haematoxylin-eosin, azan, azocarmine, and Bielschowsky and Masson’s trichromic dye (McManus & Mowry, 1968). The study was carried out using a Nikon Eclipse 80i microscope and DS camera control unit DS-L1.

The nomenclature used corresponds to the English version of the ‘Terminologica Anatomica’ (2001) of the Federative Committee on Anatomical Terminology (FCAT).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Embryonic period

Appearance and location of the interhyale (O’Rahilly stages 16 and 17)

At stage 16, the interhyale was observed for the first time as a mesenchymal condensation of the second arch situated in front of the facial nerve. At stage 17, the lower portion of the stapedius anlage was composed of two condensations separated by the stapedial artery, which formed the branches of the stapes (Fig. 1). The interhyale had a precise location, as it connected the dorsolateral condensation of the stapedial anlage, which will form the posterior limb with the cranial end of the precartilaginous mesenchymal condensation that will make up Reichert’s cartilage (Figs 1 and 2A).

image

Figure 1.  Human embryo GV-6 (13 mm CR; O’Rahilly stage 17). Haematoxylin-eosin staining. Transverse section. Location of the interhyale (I) and its relationship with the facial nerve (F). The interhyale connected with the stapedial limb (SL). Anlage of the stapedial limbs separated by the stapedial artery (SA).

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image

Figure 2.  Diagrammatic representation explaining the most important stages of the development of the stapedius muscle and of the pyramidal eminence in human embryos and fetuses. (A) O’Rahilly stages 16 and 17. Appearance and location of the interhyale (I). F, facial nerve; L, laterohyale; R, Reichert’s cartilage; S, stapes. (B) O’Rahilly stages 18 and 19. Appearance of the anlage of the belly of the stapedius muscle (SB). The anlage the belly of the stapedius muscle (SB) is located medial to the facial nerve (F). I, interhyale; L, laterohyale; R, Reichert’s cartilage; O, otic capsule; S, stapes. (C) O’Rahilly stage 22. Delimitation and angulation of the interhyale. Connection of the anlage of the belly of the stapedius muscle (SB) to the interhyale. The two segments of the interhyale are arranged in different positions: cranial, internal segment of the interhyale (II); caudal, external segment of the interhyale (EI). F, facial nerve; L, laterohyale; R, Reichert’s cartilage; O, otic capsule; S, stapes. (D) O’Rahilly stage 23. Formation of the anlage of the tendon of the stapedius muscle (II). Beginning of the regression of the external segment of the interhyale (EI). F, facial nerve; L, laterohyale; S, stapes. Belly of the stapedius muscle (SB). (E) 12–14 weeks PCd. Beginning of the mesenchymal condensation around the belly of the stapedius muscle (SB), anlage of the pyramidal eminence (PE). Decrease in the angulation between the belly (SB) and tendon (T) of the stapedius muscle. The scheme shows an opening in the anlage of the pyramidal eminence to allow a view of the belly of the stapedius muscle. F, facial nerve; L, laterohyale; S, stapes.

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Appearance of the belly anlage of the stapedius muscle (O’Rahilly stages 18 and 19)

The anlage that would give rise to the belly of the stapedius muscle was examined. It was located medial to the facial nerve and near the interhyale but independent of it (Fig. 3).

image

Figure 3.  Human embryo CIV-2 (16 mm CR; O’Rahilly stage 18). Azan staining. Transverse section. Appearance of the anlage of the belly of the stapedius muscle (SB), situated medial to the facial nerve (F). The interhyale (I) lies between the stapes (S) and the cranial end of the precartilage of the second pharyngeal arch (laterohyale; L). IN, incus; M, malleus.

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The interhyale did not undergo any structural modification but rather remained a mesenchymal condensation, though better delimited. The interhyale formed a mesenchymal bridge connecting the stapes with the cranial end of the future Reichert’s cartilage (laterohyale) (Figs 2B and 3). Both the stapes in the condrogenesis phase, as well as the precursor of Reichert’s cartilage, were clearly differentiated from the interhyale.

Interhyale differentiation. Continuity between the cranial end of Reichert’s cartilage (laterohyale) and the otic capsule (O’Rahilly stages 20 and 21)

Ossicular anlagen in the cartilaginous phase were well configured. It was observed that the crus longum of the incus established contact with the stapes and this in turn with the interhyale. The interhyale, well developed, was located between the stapes and Reichert’s cartilage and was well differentiated from both structures (Fig. 4). Laterally, the interhyale reached the area where the cranial end of Reichert’s cartilage (laterohyale) had established continuity with the lower prolongation of the otic capsule.

image

Figure 4.  Human embryo AR (21.5 mm CR; O’Rahilly stage 20). Haematoxylin-eosin staining. Frontal section. The interhyale (I) is clearly differentiated from the nearby structures. The interhyale (I) lies between the stapes (S) and the cranial portion of Reichert’s cartilage (laterohyale; L). F, facial nerve; IN, incus; M, malleus.

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Delimitation and angulation of the interhyale. Connection of the anlage of the belly of the stapedius muscle at the interhyale (O’Rahilly stage 22)

The interhyale appeared angular. The stapedius muscle was fixed at the vertex of the angulation that formed the interhyale. This morphology of the interhyale enabled the delimitation of the two segments, one cranial and a thicker internal segment, from the vertex of the angulation to the stapes, and another caudal and external from the attachment of the stapedius muscle to the zone where the cranial end of Reichert’s cartilage (laterohyale) continued with the otic capsule (Figs 2C and 5).

image

Figure 5. Human embryo GI-4 (26.5 mm CR; O’Rahilly stage 22). Azan staining. Frontal section. Delimitation and angulation of the interhyale. The anlage of the belly of the stapedius muscle (SB) is attached at the vertex of the angulation formed by the interhyale (arrow). In the interhyale is visibly delimited by two segments, internal (II) and external (EI). F, facial nerve; IN, incus; L, laterohyale; S, stapes.

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Formation of the anlage of the tendon of the stapedius muscle. Beginning of the regression of the external segment of the interhyale (O’Rahilly stage 23)

At the end of the embryonic period, the cranial end of Reichert’s cartilage (laterohyale) was clearly curved. This portion was joined by the inferolateral prolongation of the otic capsule. Between the stapes and the cranial end of Reichert’s cartilage was situated the interhyale, with two well-defined segments: one internal and thick, being the future tendon of the stapedius muscle, and the other a thin external segment and clearly in regression (Figs 2D and 6).

image

Figure 6.  Human embryo BR-4 (28 mm CR; O’Rahilly stage 23). Azocarmine staining. Frontal section. Formation of the anlage of the tendon of the stapedius muscle. Beginning of the regression of the external segment of the interhyale (EI). The internal segment of the interhyale (II) forms the anlage of the tendon of the stapedius muscle, which is clearly differentiated from the external segment that is attached to the area where the laterohyale (L) is continuous with the otic capsule (O). F, facial nerve; S, stapes.

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Fetal period

Complete regression of the lateral segment of the interhyale. Angulation between the belly and the tendon of the stapedius muscle (9–11 weeks of PCd)

At the beginning of the fetal period (9 weeks PCd) the total regression of the external segment of the interhyale had been completed. There was an angulation between the tendon, of uniform thickness, and the belly of the stapedius muscle.

At 10–11 weeks PCd, the stapedius muscle was formed by a conical belly. It was situated medial to the facial nerve, in the upper portion of the future vertical part of the facial canal and surrounded by a uniform mesenchyme that occupied the entire tympanic cavity. The vertex of the belly of the stapedius muscle was continued by the tendon, which was angled in relation to the belly. The tendon was inserted into the posterior side of the head of the cartilaginous stapes. No sign of formation of the pyramidal eminence was detected (Fig. 7A).

image

Figure 7.  (A) Human fetus VR-2 (10 weeks PCd). Azocarmine staining. Transverse section. Angulation between the belly (SB) and the tendon (T) of the stapedius muscle. F, facial nerve; I, incus; O, otic capsule; S, stapes. (B) Human fetus JR (11 weeks PCd). Azocarmine staining. Transverse section. The cranial end of Reichert’s cartilage (laterohyale; L) formed a curve surrounding the facial nerve to contribute, with the otic capsule, to the formation of the caudal portion of the vertical part of the facial canal. T, tendon of the stapedius muscle; I, incus; S, stapes.

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The cranial end of Reichert’s cartilage (laterohyale) was situated caudally with respect to the stapedius muscle and formed a curve around the facial nerve, to contribute to the formation, with the otic capsule, of the caudal portion of the vertical part of the facial canal (Fig. 7B).

Beginning of the mesenchymal condensation around the stapedius muscle, anlage of the pyramidal eminence. Decrease in the angulation between the belly of the tendon of the stapedius muscle (12–14 weeks PCd)

At 12 weeks PCd, examination revealed the way in which the condensation of the mesenchymal tissue around the belly of the stapedius muscle, the anlage of the pyramidal eminence, began. At 13 weeks PCd, the condensation of the mesenchymal tissue was more marked, and the delimitation of the anlage of the pyramidal eminence was very clear, being separated from the belly of the muscle by a constant acellular space. At 13–14 weeks PCd, the angle between the belly and the tendon of the stapedius muscle had diminished (Figs 2E and 8).

image

Figure 8.  Human fetus Be-608 (13 weeks PCd). Haematoxylin-eosin staining. Transverse section. Beginning of the condensation of mesenchymal tissue around the belly of the stapedius muscle (SB), anlage of the pyramidal eminence (PE). Decrease in the angulation between the belly (SB) and the tendon (T) of the stapedius muscle. CT, chorda tympani nerve. F, facial nerve; I, incus; M, malleus; O, otic capsule; S, stapes.

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Differentiation and delimitation of the anlage of the pyramidal eminence. Alignment between the tendon and the belly of the stapedius muscle (15–17 weeks PCd development)

An increase and differentiation was noted in the conical mesenchymal condensation that would give rise to the pyramidal eminence. This mesenchymal cover enclosed only the belly of the muscle and delimited it from the adjacent structures (Fig. 9). In section, it had a triangular shape, with a base that was attached to the posterior wall of the vertical part of the facial canal. This arrangement was similar to that of the mesenchymal tissue surrounding the distal portion of the tensor tympanic muscle. In this phase, the angulation that had existed in the belly and the tendon of the stapedius muscle in earlier stages had disappeared (Fig. 9).

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Figure 9.  Human fetus B-28 (17 weeks PCd). Azan staining. Transverse section. Delimitation and differentiation of the anlage of the pyramidal eminence (PE). Alignment of the belly (SB) and the tendon (T) of the stapedius muscle. F, facial nerve; I, incus; M, malleus; O, otic capsule; S, stapes.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

According to my observations, the stapedius muscle was formed by two anlagen: one for the tendon and another for the belly. The anlage of the tendon will be formed from a portion of the structure called the interhyale. The interhyale was detected in embryos of O’Rahilly stage 16, in the mesenchyme of the second pharyngeal arch, located between the cranial end of the first pharyngeal pouch and the facial nerve. This formation has been referred to by Hanson et al. (1962) and Louryan (1993), while Rodríguez-Vázquez (2005) analysed the initial stages, establishing the independent origin of Reichert’s cartilage, as well as the relationship that the interhyale had with the stapedial anlage. However, until now, there had been no study of all its developmental phases.

The anlage of the belly of the stapedius muscle was observed at O’Rahilly stage 18 at the cranial end of the mesenchyme of the second pharyngeal arch, medial to the facial nerve and near the interhyale, but forming a completely independent anlage. These data are consistent with the hypothesis that all cranial myoblasts would derive from the paraxial mesoderm (demonstrated in the bird by Noden et al. 1999).

During O’Rahilly stages 18 and 19, the interhyale underwent no structural modification and continued to remain as a well-defined mesenchymal condensation which connected the stapes to the cranial end of Reichert’s cartilage. Therefore, I disagree with Anson & Bast (1946), in that the stapes had a direct cartilaginous continuity with Reichert’s cartilage and that when this continuity was lost, it would remain joined by the hyostapedial connection. Also, I disagree with the authors (Anson & Cauldwell 1942; Anson et al. 1944; Anson & Bast 1946) who contended that the stapedius muscle would be formed from the hyostapedial connection and with those (Hanson et al. 1962; Louryan, 1993) who indicated that it would be formed from the interhyale. According to my analysis, the hyostapedial connection and the interhyale correspond to the same structure.

Reichert’s cartilage, in my opinion, is not bifurcated in two structures: the interhyale and the laterohyale as remarked by Hanson et al. (1962). According to my observations, the cranial end of Reichert’s cartilage presents a curved morphology and is oriented laterally, to continue from O’Rahilly stage 20, with the lower and lateral prolongation of the otic capsule, which was pointed out by Macklin (1921) as the crista parotica. This part of Reichert’s cartilage corresponds to the laterohyale, as indicated by Rodríguez-Vázquez et al. (2006). Previous works have studied ossicle development in the mouse (Louryan, 1986) and showed that the mouse laterohyale is very similar to the human one. In my view, this arrangement and morphology is key to understanding the underlying processes of development in this region.

At O’Rahilly stage 22, two facts of great importance were noted: first, the clear connection between the interhyale and the anlage of the belly of the stapedius muscle and, secondly, a morphological change in the interhyale. This latter structure altered its morphology, forming an angle that Rodríguez-Vázquez (2005) defined at this stage as the delimitation of the interhyale. The anlage of the belly of the stapedius muscle established a connection with the interhyale, attaching itself to the vertex of the angulation. Therefore, two segments would be delimited in the interhyale – one thicker, internal, from the vertex of the angulation to the anlage of the stapes, and another external segment from the attachment of the belly of the stapedius muscle to the zone where the cranial end of Reichert’s cartilage (laterohyale) is continued with the otic capsule.

The internal segment of the interhyale, at O’Rahilly stage 23, would form the anlage of the tendon of the stapedius muscle. On the contrary, the external segment of the interhyale began its regression to finally disappear completely, without leaving any vestiges in the first stages of the fetal period. This arrangement, which I observed in this specific study on the development of the stapedius muscle, offers a new vision and clarifies the development and formation of the stapedius muscle. It contradicts the classical conception in which this structure was thought to derive completely from the hyostapedial connection (Anson & Cauldwell, 1942; Anson et al. 1944; Anson & Bast, 1946) or the interhyale one (Hanson et al. 1962). Also, this new evidence contradicts those authors that theorized without making embryonic studies, believing that it derived from the remains of a second visceral bar (Hough, 1958; Grant & Grant, 1991; Patel, 1972).

An observation made by no other author is the marked angulation we found existing in the tendon and belly of the stapedius muscle. This angulation, visible from the formation of the tendon of the stapedius muscle diminishes progressively over the fetal period until its alignment between 15–17 weeks PCd.

In agreement with my observations, the pyramidal eminence would be formed from mesenchymal tissue of the tympanic cavity. From 12 weeks PCd, this mesenchymal tissue is condensed around the belly of the stapedius muscle, constituting the anlage of the pyramidal eminence. This arrangement is similar to that which existed surrounding the distal portion of the tensor tympani muscle. Between weeks 15–17 PCd, greater differentiation and delimitation was observed in the condensation of the mesenchymal anlage of the pyramidal eminence, as well as the alignment of the tendon and the belly. These observations have not been reported by any other author. Therefore, I cannot agree that the pyramidal eminence is formed from the laterohyale, as proposed by Hanson et al. (1962), as I was able to confirm clearly that the laterohyale was joined and continued with the inferolateral prolongation of the otic capsule, to form together the most caudal portion of the vertical part of the facial canal. Therefore, the laterohyale presented another location, without any relationship to the belly of the stapedius muscle. Nor should the hypothesis be accepted that this structure derived from pre-cartilaginous cells of the second pharyngeal arch (Hough, 1963; Patel, 1972; Grant & Grant, 1991).

The great variations in the length of the tendon of the stapedius muscle identified in adults by Hough (1958) and Cremers & Hoogland (1986) could be explained depending on the place of fixation of the belly of the stapedius muscle in the interhyale, which would determine the length of the internal segment (anlage of the tendon) and consequently the length of the tendon of the stapedius muscle. In addition, the persistence of a greater or lesser degree of angulation observed between the tendon and the belly of the stapedius muscle would explain the variations in the direction of the insertion and the different positions in which the tendon can be presented, as indicated by Hough (1958) and Patel (1972).

The development of the stapedius muscle, in two different anlagen, observed in this study, explains coherently and logically the very rare anomalies described by Kelemen (1943) and Marquet (1981), in which the absence of the tendon is associated with the belly of the muscle developed. Nandapalan & Tos (2000) tried to explain this anomaly, basing their argument on the hypothesis of Hough (1963), by stating that the tendon and the muscle (apparently referring to the belly of the stapedius muscle) must have two different origins. Nevertheless, the formulation of these theories without the support of embryological studies is erroneous as they begin from the assumption that the entire interhyale would give rise to the tendon and that the pyramidal eminence would be formed from the laterohyale. From my perspective, the embryological interpretations presented up to now to explain the variations and anomalies of this region have been confusing and partly or entirely erroneous. These theories should be reviewed and should not be used as the basis to explain the variations and anomalies of this region.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
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